PROJECT SUMMARY/ABSTRACT Our OVERALL OBJECTIVE is to elucidate the molecular basis of activation of class B GPCRs, using the prototypic secretin receptor (SecR) as a model. Insights will fill key gaps in knowledge and facilitate ultimate development of drugs exhibiting various activity profiles. Class B GPCRs include established targets for treatment of diabetes, obesity, osteoporosis, migraine, anxiety, and depression. However, therapeutics remain suboptimal, and the receptor class has been refractory to development of small molecule, orally active drugs, at least in part, due to lack of understanding of the structure and functional dynamics required for receptor activation. We now have novel, unique insights into both of these, including high-resolution structures of related, full-length, G protein-coupled holoreceptors, that highlight the importance of the interface between the receptor N-terminal extracellular domain (ECD) and the transmembane domain core in ligand binding and receptor activation. Component aims are directed toward understanding the conformational dynamics of this interface for agonist binding and receptor activation, and using our breakthroughs in use of single particle cryo- EM to provide a structural framework for this work. Aim 1, elucidates molecular events involved in secretin peptide engagement with its receptor core, and key determinants for its activity, testing the hypothesis that orientation (and interaction) of ECD and core domains plays a critical role in directing and positioning the orthosteric agonist pharmacophore near its site of action. We will explore this locus using cysteine trapping to compare spatial approximations for analogous inactive and active probes, applied to wild type receptor, as well as dimerization-deficient receptor constructs. Rational structure-activity analysis for binding and a broad range of biological activities of the agonist pharmacophore will also be performed, with results used to provide insights into determinants of activation and effector specificity. Aim 2, investigates the relative orientations and interactions between SecR ECD and core, exploring functional implications of this interrelationship, testing the hypothesis that these domains can interact in various ways that affect states of quiescence and activation. This will be approached by receptor mutagenesis to modify domain interactions, establishment of domain-domain disulfide bonds by incorporating cysteines at the top of the receptor core and bottom of the ECD, as well as using immunologic probes of predicted surfaces of the receptor amino terminus to determine access and to be used in resonance transfer techniques. Aim 3, elucidates SecR inactive and active/holostructure using single particle cryo-EM, testing the hypothesis that mapping of biochemical and functional data onto high resolution 3D structures, including agonist-receptor-heterotrimeric G protein and inactive antagonist-occupied receptor, will help to elucidate the molecular basis for receptor signaling. Together, this work will provide fundamental advances in understanding of class B GPCR structure and function, with insights highly useful in drug development targeting these receptors.